Assessment of microbial contamination in laser materials processing laboratories used for prototyping of biomedical devices.

Microbial contamination of medical devices during pilot production can be a significant barrier as the laboratory environment is a source of contamination. There is limited information on microbial contaminants in laser laboratories and environments involved in the pilot production of medical devices. This study aimed to determine the bioburden and microbial contaminants present in three laser laboratories - an ISO class 7 clean room, a pilot line facility and a standard laser laboratory. Microbiological air sampling was by passive air sampling using settle plates and the identity of isolates was confirmed by DNA sequencing. Particulate matter was analysed using a portable optical particle counter. Twenty bacterial and 16 fungal genera were isolated, with the genera Staphylococcus and Micrococcus being predominant. Most isolates are associated with skin, mouth, or upper respiratory tract. There was no significant correlation between microbial count and PM2.5 concentration in the three laboratories. There were low levels but diverse microbial population in the laser-processing environments. Pathogenic bacteria such as Acinetobacter baumannii and Candida parapsilosis were isolated in those environments. These results provide data that will be useful for developing a contamination control plan for controlling microbial contamination and facilitating advanced manufacturing of laser-based pilot production of medical devices.

Femtosecond Laser-Induced Crystallization of Amorphous Silicon Thin Films under a Thin Molybdenum Layer.

A new process to crystallize amorphous silicon without melting and the generation of excessive heating of nearby components is presented. We propose the addition of a molybdenum layer to improve the quality of the laser-induced crystallization over that achieved by direct irradiation of silicon alone. The advantages are that it allows the control of crystallite size by varying the applied fluence of a near-infrared femtosecond laser. It offers two fluence regimes for nanocrystallization and polycrystallization with small and large crystallite sizes, respectively. The high repetition rate of the compact femtosecond laser source enables high-quality crystallization over large areas. In this proposed method, a multilayer structure is irradiated with a single femtosecond laser pulse. The multilayer structure includes a substrate, a target amorphous Si layer coated with an additional molybdenum thin film. The Si layer is crystallized by irradiating the Mo layer at different fluence regimes. The transfer of energy from the irradiated Mo layer to the Si film causes the crystallization of amorphous Si at low temperatures (∼700 K). Numerical simulations were carried out to estimate the electron and lattice temperatures for different fluence regimes using a two-temperature model. The roles of direct phonon transport and inelastic electron scattering at the Mo-Si interface were considered in the transfer of energy from the Mo to the Si film. The simulations confirm the experimental evidence that amorphous Si was crystallized in an all-solid-state process at temperatures lower than the melting point of Si, which is consistent with the results from transmission electron microscopy (TEM) and Raman. The formation of crystallized Si with controlled crystallite size after laser treatment can lead to longer mean free paths for carriers and increased electrical conductivity.

Femtosecond Laser Assisted Crystallization of Gold Thin Films.

We propose a novel low temperature annealing method for selective crystallization of gold thin films. Our method is based on a non-melt process using highly overlapped ultrashort laser pulses at a fluence below the damage threshold. Three different wavelengths of a femtosecond laser with the fundamental (1030 nm), second (515 nm) and third (343 nm) harmonic are used to crystallize 18-nm and 39-nm thick room temperature deposited gold thin films on a quartz substrate. Comparison of laser wavelengths confirms that improvements in electrical conductivity up to 40% are achievable for 18-nm gold film when treated with the 515-nm laser, and the 343-nm laser was found to be more effective in crystallizing 39-nm gold films with 29% improvement in the crystallinity. A two-temperature model provides an insight into ultrashort laser interactions with gold thin films and predicts that applied fluence was insufficient to cause melting of gold films. The simulation results suggest that non-equilibrium energy transfer between electrons and lattice leads to a solid-state and melt-free crystallization process. The proposed low fluence femtosecond laser processing method offers a possible solution for a melt-free thin film crystallization for wide industrial applications.

Laser-Induced Periodic Surface Structure Enhances Neuroelectrode Charge Transfer Capabilities and Modulates Astrocyte Function.

The brain machine interface (BMI) describes a group of technologies capable of communicating with excitable nervous tissue within the central nervous system (CNS). BMIs have seen major advances in recent years, but these advances have been impeded because of a temporal deterioration in the signal to noise ratio of recording electrodes following insertion into the CNS. This deterioration has been attributed to an intrinsic host tissue response, namely, reactive gliosis, which involves a complex series of immune mediators, resulting in implant encapsulation via the synthesis of pro-inflammatory signaling molecules and the recruitment of glial cells. There is a clinical need to reduce tissue encapsulation in situ and improve long-term neuroelectrode functionality. Physical modification of the electrode surface at the nanoscale could satisfy these requirements by integrating electrochemical and topographical signals to modulate neural cell behavior. In this study, commercially available platinum iridium (Pt/Ir) microelectrode probes were nanotopographically functionalized using femto/picosecond laser processing to generate laser-induced periodic surface structures (LIPSS). Three different topographies and their physical properties were assessed by scanning electron microscopy and atomic force microscopy. The electrochemical properties of these interfaces were investigated using electrochemical impedance spectroscopy and cyclic voltammetry. The in vitro response of mixed cortical cultures (embryonic rat E14/E17) was subsequently assessed by confocal microscopy, ELISA, and multiplex protein array analysis. Overall LIPSS features improved the electrochemical properties of the electrodes, promoted cell alignment, and modulated the expression of multiple ion channels involved in key neuronal functions.

Monitoring of pH and temperature of neuropathic diabetic and nondiabetic foot ulcers for 12 weeks: An observational study.

Wound bed assessment is largely reliant on subjective interpretation without recourse to objective tools or biomarkers. The identification of a point of care, reliable biomarker would enhance assessment and ultimately clinical decision making. Two potentially emerging wound biomarkers exist: surface pH and surface temperature. To date, knowledge of their use has been predominantly in wound prevention, in vitro studies and single time measurements. Our objective was to determine surface pH, size, and surface temperature in noninfected, neuropathic foot ulcers at baseline and at 12 weeks. 50 patients (68% [n = 34] had diabetes) participated. Mean baseline pH of wounds was 6.95 (SD 1.01); temperature 30.91 °C (SD 3.00); and size 0.82 cm2 (SD 0.61). After 12 weeks, 26% (n = 13) were lost to follow-up, 50% (n = 25) had healed. Of the remaining patients, mean pH was 6.72 (SD 0.54); temperature 30.88 °C (SD 2.97), and size 0.13 cm2 (SD 0.13). We have provided baseline values for pH and temperature of noninfected, neuropathic diabetic, and nondiabetic foot ulceration. Further studies in a larger cohort are warranted to determine if temperature and or pH are indicative of a healing or nonhealing state.

Enhanced thermal imaging of wound tissue for better clinical decision making.

Infrared cameras are increasingly applied in clinical applications as they allow fast, inexpensive and non-contact temperature measurements. As abnormal heat distribution can indicate illness, infrared cameras have been applied in the prediction, diagnosis and monitoring of medical conditions. Current practices, however, often overlook the importance of emissivity when taking thermal measurements. The consensus is that human skin has an emissivity of 0.98 but this value varies between individuals, areas examined, and if the skin is damaged. In particular, further research should be conducted on the emissivity variations of wounds. OBJECTIVE: This research investigated the emissivity variation of chronic wounds and its effect on thermal measurements. Eleven patients with non-infected foot ulcers were recruited. Three non-diabetic wounds were also investigated in a clinical setting. APPROACH: A reflectance based method was used which involved alternating shades at different temperatures over the region of interest. Based on the change in the thermal images, emissivity was calculated at each pixel. MAIN RESULTS: Overall, it was found that the emissivity of wounds was similar or slightly higher to intact skin (range 0.01-0.03 higher with an average value of 0.97 ± 0.03), with lower values at wound edges (on average 0.02 lower than intact skin). Correcting for emissivity resulted in an average temperature difference of 0.83% in the thermal images. SIGNIFICANCE: Despite the similarity in emissivity, the difference between the original thermal image and the emissivity corrected thermal image in some cases was substantial. These differences could prove significant in clinical evaluations, indicating the need to incorporate emissivity measurement into standard protocol to ensure utmost accuracy.

Laser microfabrication of a microheater chip for cell culture outside a cell incubator.

Microfluidic chips have demonstrated their significant application potentials in microbiological processing and chemical reactions, with the goal of developing monolithic and compact chip-sized multifunctional systems. Heat generation and thermal control are critical in some of the biochemical processes. The paper presents a laser direct-write technique for rapid prototyping and manufacturing of microheater chips and its applicability for lab-on-a-chip cell culture outside a cell incubator. The aim of the microheater is to take the role of conventional incubators for cell culture for facilitating microscopic observation and/or other online monitoring activities during cell culture and provides portability of cell culture operation. Microheaters (5mm×5mm) have been successfully fabricated on soda-lime glass substrates covered with aluminium layer of thickness 120nm. Experimental results show that the microheaters exhibit good performance in temperature rise and decay characteristics, with localized heating at targeted spatial domains. These microheaters were suitable for a maximum long-term operation temperature of 120°C and validated for operation at 37°C for 48h. Results demonstrated that the microheaters are suitable for the culture of immortalised cell lines. The growth and viability of SW480 colon adenocarcinoma cells cultured the developed microheater chip were comparable to the results obtained in a conventional cell incubator.

In one harness: the interplay of cellular responses and subsequent cell fate after quantum dot uptake.

Rapid growth and expansion of engineered nanomaterials will occur when the technology can be used safely. Quantum dots have excellent prospects in clinical applications, but the issue of toxicity has not yet been resolved. To enable their medical implementation, the effect on, and mechanisms in, live cells should be clearly known and predicted. A massive amount of experimental data dedicated to nanotoxicity has been accumulated to-date, but it lacks a logical structure. The current challenge is to organize existing knowledge into lucid biological and mathematical models. In our review we aim to describe the interplay of various cell death mechanisms triggered by quantum dots as a consequence of particle parameters and experimental conditions.

Comparison of ablation mechanisms at low fluence for ultrashort and short-pulse laser exposure of very thin molybdenum films on glass.

Complete removal of a loosely adhered very thin molybdenum film from a glass substrate is investigated for both femtosecond and nanosecond lasers at different wavelengths. Atomic force microscopy and scanning electron microscopy confirm that ablation of the molybdenum film by femtosecond pulses occurs close to the damage threshold fluence, creating minimal damage to the substrate. This is in contrast to nanosecond laser processing where significant substrate damage at the equivalent damage threshold fluence is observed. Simulations predict a two-stage mechanical buckling mechanism in the femtosecond case. Out-of-plane thermal expansion first results in a tensile expansion of molybdenum film from the glass substrate; this locally delaminated film is then buckled by a subsequent compressive stress, leading to thin film spallation. Ablation by nanosecond laser pulses behaves differently. The appreciable heat diffusion length (∼700 nm) in molybdenum, observed for the nanosecond case, results in an increased thermal expansion of the glass. The thermally induced stress generated by the molten glass creates a delaminated area, which "pushes" the compressed film away from the substrate. These findings are relevant to future selective laser patterning of very thin molybdenum layers.

Sensing of p53 and EGFR Biomarkers Using High Efficiency SERS Substrates.

In this paper we describe a method for the determination of protein concentration using Surface Enhanced Raman Resonance Scattering (SERRS) immunoassays. We use two different Raman active linkers, 4-aminothiophenol and 6-mercaptopurine, to bind to a high sensitivity SERS substrate and investigate the influence of varying concentrations of p53 and EGFR on the Raman spectra. Perturbations in the spectra are due to the influence of protein-antibody binding on Raman linker molecules and are attributed to small changes in localised mechanical stress, which are enhanced by SERRS. These influences are greatest for peaks due to the C-S functional group and the Full Width Half Maximum (FWHM) was found to be inversely proportional to protein concentration.

Single-pulse laser ablation threshold of borosilicate, fused silica, sapphire, and soda-lime glass for pulse widths of 500 fs, 10 ps, 20 ns.

In this work, we report a comparative study of the laser ablation threshold of borosilicate, fused silica, sapphire, and soda-lime glass as a function of the pulse width and for IR laser wavelengths. We determine the ablation threshold for three different pulse durations: τ=500 fs, 10 ps, and 20 ns. Experiments have been performed using a single laser pulse per shot in an ambient (air) environment. The results show a significant difference, of two orders of magnitude, between the group of ablation thresholds obtained for femtosecond, picosecond, and nanosecond pulses. This difference is reduced to 1 order of magnitude in the soda-lime substrate with tin impurities, pointing out the importance of the incubation effect. The morphology of the marks generated over the different glass materials by one single pulse of different pulse durations has been analyzed using a scanning electron microscope (FESEM ULTRA Plus). Our results are important for practical purposes, providing the ablation threshold data of four commonly used substrates at three different pulse durations in the infrared regime (1030-1064 nm) and complete data for increasing the understanding of the differences in the mechanism's leading ablation in the nanosecond, picosecond, and femtosecond regimes.

Mechanically inspired laser scribing of thin flexible glass.

Laser cutting of thin glass (<100 µm) has proven problematic. We describe an alternative laser scribing method that utilizes surface stress raisers. An ultrashort laser source is used to precisely pattern a plurality of aligned elliptical recesses on the glass. The apex of an ellipse concentrates applied tensile stresses. Depending on the elliptical dimensions, the stress concentration factor can be several tens of magnitude. The orientation of the ellipses defines a preferred scribing path. Tensile stress is applied orthogonally to the path and causes mode I fracture. The resulting scribe is of higher quality and strength than are possible with a full body laser cut. The optical setup is simple, low in cost, and compatible with future roll-to-roll manufacturing. The stress field around a stress raiser was analyzed using finite element method analysis. Consequently, the stress raiser process offers an alternative to other processes which employ high numerical aperture optics for thin glass scribing.

Laser direct-write technique for fabricating microlens arrays on soda-lime glass with a Nd:YVO4 laser.

A one-step direct-write technique for fabricating spherical microlenses on soda-lime glass substrates is described. Using a Q switched Nd:YVO(4) laser combined with a galvanometer system, square and triangular microlens arrays were fabricated. The focal length of microlenses is measured using direct and nondirect methods. Values around 118 and 125 µm were obtained for the microlens focal length of square and triangular arrays, respectively. A noncontact profilometer is used for determining the surface roughness of square and triangular arrays. Results are compared with that of glass substrate.